Method for the visual detection of specific antibodies by the use of lateral flow assays

ABSTRACT

This disclosure teaches a method and composition for detecting the presence of class specific antibodies reactive with analytes such as bacteria, allergens, autoimmune antigens, viral proteins, haptens, and carbohydrates by lateral flow techniques. In one embodiment of the disclosure, a test sample is added to the test strip, which then migrates to the site of the immobilized allergens, thereby forming a first antibody IgE-allergen complex. A chase release buffer is added upstream to or from the site of the labeled anti-IgE antibodies, which is itself upstream from the sample site. The anti-IgE antibodies migrate downstream to the site of immobilized first complex, thereby forming a second complex indicating the presence of class specific IgE antibodies in the test specimen. In another embodiment of the disclosure, a liquid form of the labeled anti-IgE antibodies can be added to the test strip after the first complex has been formed. 
     These techniques can be used to perform other lateral flow tests.

This application is a divisional of U.S. patent application Ser. No.11/038,076, filed Jan. 21, 2005, incorporated herein by reference.

BACKGROUND OF THE DISCLOSURE

1. Field of the Disclosure

The present disclosure discloses a method and composition for detectingthe presence of antibodies in human or animal bodily fluids (blood,serum, plasma, urine, colostrum, milk, tears, or saliva) to analytessuch as bacteria, Chlamydiae, Rickettsiae, protozoa, allergens,autoimmune antigens, viral proteins, and carbohydrates by lateral flowtechniques.

2. Description of the Prior Art

Over the years, numerous patents have been issued involvingimmuno-chromatographic devices. The standard features of these devicescomprise the following:

a) A plastic or paper housing allowing the viewing of a reaction area ona bibulous (lateral flow) strip;

) an opening at one end of the housing allowing for the addition ofsample (urine, blood, plasma, serum or bacteria in a media base);

c) Bibulous material (the lateral flow strip) having immobilizedspecific binding members (analytes) capable of reacting with antigens orantibodies.

d) A pad of absorbent bibulous material (the absorbent pad) enclosed atthe end opposite the sample well and used to absorb transversely flowingsample, buffers and colloids;

e) A strip of bibulous material used in the sample well end to initiallyabsorb the sample being applied;

f) A strip of bibulous material in contact with the sample well materialand the lateral flow strip and containing a dried colored solid phasereagent, the solid phase coated with proteins or haptens.

Two types of chromatographic immunoassays are commonly described. In theone, proteins, haptens, or small molecule analytes contained in bodilyfluids (urine, blood, plasma, serum, and saliva) are detected. Theanalytes include hCG, FSH, LH, CKMB, TSH, troponins, myoglobulin, cancerproteins, viralbacterial proteins, haptens, therapeutic drugs, and drugsof abuse.

In the other chromatographic immunoassay, the analyte being detected is(are) human antibody (antibodies) of various classes specificallyreactive with agents such as viral or bacterial proteins (HIV, HepatitisA and C, H. pylori, EBV, Rubella, CMV, HSV, Dengue fever, Lyme, Chagas,TB, Toxoplasma, autoimmune antigens, etc.) or allergens (pollens, molds,dust/mites, foods, animal epithelia, etc.). When it comes to detectingantibody, three formats are typically used:

1) The colored solid phase [SP] is coated with proteins or lectins[protein A, protein G, lentil lectin, jacalin, concanavilin A, mannanbinding protein, wheat germ lectin, peanut lectin and avidchrom] thatreact with human IgG antibodies. The solid phase may be coated withanti-immunoglobulins that specifically react with IgG, IgM, IgA, or IgEcontained in the sample to be analyzed. The bibulous strip would in thiscase contain the analyte of interest to which the specific antibodycontained in the sample reacts.

2) The colored solid phase contains the analyte to which the humanimmunoglobulins react. The bibulous strip would in this case alsocontain the analyte of interest to which the specific antibody containedin the sample reacts.

3) The colored solid phase contains the analyte to which immunoglobulinsreact. The bibulous strip contains proteins directed against variousclasses of immunoglobulins or substances such as protein A, protein G,lectins, lentil lectin, jacalin, concanavilin A, mannan binding protein,wheat germ lectin, peanut lectin and avidchrom or a mix of antibody toimmunoglobulin classes IgG, IgA, IgM and IgE.

U.S. Pat. No. 5,459,041 (Blaser et al.) discloses antigenic compositionsfor use in diagnostic kits and the like for detecting the presence ofantibodies specific for Campylobacter pylori, Samples of bodily fluids,for instance, may be contacted with immobilized antigen on a solid phasewhich is then washed and tested for the occurrence of significant levelsof antigen/antibody complex. Levels exceeding a predetermined positivethreshold are indicative of antibodies to Campylobacter pylori in thesample tested. Kits employing the antigenic compositions of thedisclosure preferably include means for detecting the antigen/antibodycomplex such as materials and reagents for conducting an enzyme-linkedimmunosorbent assay, Western blot technique,

ELISA, liposome-based assay or other known detection tests. The Westernblot and ELISA tests used here are for the detection of IgA and IgGantibodies.

U.S. Pat. No. 5,567,594 (Calenoff) discloses a library of isolated andpurified antigens specific for a microorganism is a set of individualmolecules. The library forms antigen-antibody complexes useful in thecontext of diagnosing and treating conditions associated with a specificmicroorganism such as H. pylori-induced gastro-duodenal disease.Antigen-antibody complexes with IgA, IgG and IgM are also useful if theantigen is a bacteria. By this multi variate approach, a specificcondition is diagnosed with high sensitivity and specificity bydetermining whether complexes form between a specific antigen libraryand a biological sample which contains immunoglobulins from anindividual. Such libraries also are useful for immunotherapy. Westernblot is used to detect IgE antibodies. The method requires enzymeconjugates and enzyme substrates and two wash steps to detectantibodies.

U.S. Pat. No. 5,420,014 (Cripps et al.) discloses a method for detectinga current infection by H. pylori in a mammal. The method comprisescontacting a mucous secretion [saliva] from said mammal with animmobilized antigen component from H. pylori for a time and underconditions sufficient for an IgG antibody in said mucous secretionspecific to a antigen component to form a complex therewith and thensubjecting said complex to a detecting means which involves an enzymeconjugate and specific substrate.

U.S. Pat. No. 6,068,985 (Cripps) discloses a method which uses saliva todetect IgG in both the Western Blot and ELISA tests. This detectionmethod requires the use of an enzyme conjugate and enzyme substrate andtwo wash steps to detect the antibody.

U.S. Pat. No. 5,846,751 (Pronovost et al.) discloses a sensitive andspecific antigen preparation for the detection of Helicobacter pylori inbiological samples. The preparation uses a range of antigens derivedfrom size exclusion chromatography of detergent-solubilized H. pyloricells and the purified antigen preparation is coated on the solid phase.Serological assays such as ELISA, latex agglutination, and rapid EIAassays are used to detect antibodies to H. pylori. The disclosure alsouses a lateral flow device to detect total immunoglobulins to H. pylori.In this case, the H. Pylori antigen is striped on the membrane reactionarea and also coated to the colored solid phase. The antibody in thesample reacts first with H. pylori gold coated conjugate, and thentravels to the membrane reaction area where it reacts with striped H.pylori.

U.S. Pat. No. 5,200,344 (Blaser et al) uses a purified p28 kd proteinfrom H. pylori to detect IgA, IgM and IgG antibody in ELISA and WesternBlot. The test requires conjugate and enzyme substrate and two washsteps to detect the antibody.

U.S. Pat. Nos. 6,060,326 and 5,945,294 (Frank et al.) discloses methodsto detect canine IgE using a canine Fc epsilon receptor to detect canineIgE antibodies in a biological sample from a canine.

U.S. Pat. No. 5,547,833 (Dorval et al.) discloses a radial flow assaydelivery device, and methods of use.

U.S. Pat. No. 5,622,871 (May et al.) discloses an analytical test deviceuseful for example in pregnancy testing, includes a hollow casingconstructed of moisture-impervious solid material, such as plasticsmaterials, containing a dry porous carrier which communicates indirectlywith the exterior of the casing via a bibulous sample receiving memberwhich protrudes from the casing such that a liquid test sample can beapplied to the receiving member and permeate therefrom to the porouscarrier, the carrier containing in a first zone a labeled specificbinding reagent is freely mobile within the porous carrier when in themoist state, and in a second zone spatially distinct from the first zoneunlabeled specific binding reagent for the same analyte which unlabeledreagent is permanently immobilized on the carrier material and istherefore not mobile in the moist state, the two zones being arrangedsuch that liquid sample applied to the porous carrier can permeate viathe first zone into the second zone, and the device incorporating anaperture in the casing, enabling the extent (if any) to which thelabeled reagent becomes bound in the second zone to be observed.Preferably the device includes a removable cap for the protrudingbibulous member. Additionally, May teaches that all of the reagents,analyte reactions, and complexes occur within a single test strip.

U.S. Pat. No. 6,485,982 (Charlton) discloses a test cell and a methodfor detection of a preselected ligand in a liquid sample such as a bodyfluid. The test cell includes an elongate outer casing which houses aninterior permeable material capable of transporting an aqueous solutionand defining a sample inlet, a test volume, and a reservoir volume. Thereservoir volume is disposed in a section of the test cell spaced apartfrom the inlet and is filled with sorbent material. The reservoir actsto receive liquid transported along a flow path defined by the permeablematerial and extending from the inlet and through the test volume. Inthe test volume is a test site which includes a first protein having abinding site specific to a first epitope of the ligand immobilized influid communication with the flow path. The test site can be observedthrough a window of the casing. Like May, this patent teaches that allof the reagents, analyte reactions, and complexes occur within a singletest strip.

U.S. Pat. No. 6,528,325 (Hubscher et al.) discloses a method andcomposition for detecting the presence of class specific antibodiesreactive with analytes such as bacteria, allergens, autoimmune antigens,viral proteins, and carbohydrates by lateral flow techniques. In oneembodiment of the invention, a test sample obtained from bodily fluidsreacts with a gold labeled antigen. The resulting complex travels acrossthe membrane, and along the lateral flow strip. Red colored lines formedin specific locations along the test strip where anti-class specificantibodies have been immobilized indicate the presence of class specificantibodies in the test specimen. In another embodiment of the invention,the lateral flow assay serves as an immunochromatographic screening testfor the detection of allergen-specific IgE antibodies in human serum.Test sample reacts with gold labeled anti-IgE antibody. The resultingcomplex travels across the membrane where immobilized allergens capturethe allergen specific IgE-anti-IgE complex. Colored lines are formed inthe test areas to indicate the presence of allergen-specific IgEantibodies.

SUMMARY OF THE DISCLOSURE

The proposed disclosure allows for a greatly improved antibody classrecognition. In one embodiment of the disclosure, a lateral flowimmunoassay device distinguishes at least three classes of antibody. Theclasses of antibody to be distinguished include IgG, IgA and IgM. Acontrol line reactive with gold particles is also present. The newarrangement on the test strip of the lateral flow assay of the presentdisclosure greatly improves accuracy and readability.

Specifically, the lateral flow immunoassay device for detecting immunereactants, comprises at least one test strip. Each test strip has asample site for applying a sample comprising antibodies. There are aplurality of reaction sites downstream from the sample site, with eachsaid reaction site containing a different allergen such that when IgEantibodies come in contact with an antigen to which the IgE antibodiesreact, a first complex between the sample IgE and the immobilizedallergen is formed. In contrast to the prior art colorimetric labeledanalyte site comprising a colorimetric labeled anti-IgE antibody, ispositioned upstream from said sample site. There is a location on saidtest strip to add a chase release buffer for the release of the labeledanalyte. With the addition of said chase release buffer the labeledanalyte (gold labeled anti-IgE) migrates to the site of the at least onefirst complex, and a colorimetric labeled Anti-IgE antibody- antibodyIgE-allergen-complex is formed. This novel arrangement greatly improvesthe accuracy and readability of positive results

In another embodiment of the disclosure, the immunoassay test strip ismodified to allow detection of the IgE class of antibody to the manyallergens coated sequentially on a bibulous strip.

In another embodiment of the disclosure, IgG reacting protein (which canbe protein A, protein G, an antibody to IgG or lectins such as lentillectin, jacalin, concanavilin A, mannan binding protein, wheat germlectin, peanut lectin and avidchrom) is added to the sample pad in orderto complex the IgG contained in the sample such that the molecularweight of the IgG complex is greater than 1.0 million. This largecomplex travels sufficiently slower than IgA, IgM, and IgE therebyallowing these antibodies to react prior to the IgG. The various reactedcomplexes are captured on the bibulous strip coated at three sites withantibody to IgM, IgA and IgG or a protein/lectin reactive with IgG(protein A, protein G, lentil lectin, jacalin, concanavilin A, mannanbinding protein, wheat germ lectin, peanut lectin and avidchrom). Thus,the class of reactive antibody is distinguished.

In one embodiment of the disclosure, all of the reagents, including thecolored labels, the reaction sites, and the site at which the sample isadded, are all positioned in the same plane within the same test strip.In this embodiment of the disclosure, for example, the colored labelsmay be embedded or absorbed within the sorbent material of the teststrip.

In another embodiment of the disclosure, at least one of the reagents,including the colored labels, the reaction sites, and the site at whichthe sample is added, are placed on a pad or site that resides on top ofsaid test strip.

In another embodiment of the disclosure, the sample site is positionedbetween the labeling site and the first binding site.

In yet another embodiment of the disclosure, the sample being tested maybe an antigen or analyte, or some other biological or chemicalsubstance, and the immobilized

BRIEF DESCRIPTION OF THE DRAWINGS

The disclosure will be better understood and further objects,characterizing features, details and advantages thereof will appear moreclearly as the following description proceeds with reference to theaccompanying diagrammatic drawings given by way of non limiting exampleonly illustrating a presently preferred specific embodiment of thedisclosure.

FIG. 1 is a perspective view of one embodiment of the lateral flowimmunoassay test;

FIG. 2 is a perspective view of another embodiment of the lateral flowimmunoassay test;

FIG. 3 is an exploded view of the lateral flow immunoassay test;

FIG. 4 is a side view of the test strip;

FIG. 5 is a view of the colored particles attached to the antigen;

FIG. 6 is an exploded view of another version of the lateral flowimmunoassay test;

FIG. 7 is a side view of yet another version of the lateral flow teststrip;

FIG. 8 is a schematic of the reactions taking place in anotherembodiment of the disclosure;

FIG. 9 is a perspective view of an embodiment of the lateral flow teststrip;

FIG. 10 is a perspective view of another embodiment of the lateral flowtest strip;

FIG. 11 is a perspective view of an another alternative embodiment ofthe invention;

FIG. 12 is a cross sectional side view of the alternative embodiment ofthe invention of FIG. 12 and

FIG. 13 is cross sectional side view of yet another embodiment of theinvention;

FIG. 14 is an overhead view of another embodiment of the invention; and

FIG. 15 is a frontal view of another embodiment of the invention.

DETAILED DESCRIPTION OF THE DISCLOSURE

The immunoassay lateral flow test system 1 comprises a casing 2,preferably plastic, surrounding a test strip 7. On the top surface ofthe test strip there is a sample opening 8 and an opening 30 for a chaserelease buffer (explained supra and infra). A test results opening 9 toshow the results of the assay. The casing can take a number of differentforms. In FIGS. 3 and 6, the casing has a top section 4 or 100, and abottom section 5 or 101.

Within the top section 4 there is at least one channel 6 into which isfitted a test strip 7. The test strip 7 preferably has a membranesupport 10. The membrane support 10 may be comprised of plastic,cardboard, or any other rigid material. On top of the membrane support10 is a testing layer 11, preferably made out of nitrocellulose. On topof the nitrocellulose or testing layer 11 are the areas to which theappropriate reagents or antigens are applied or affixed. Thenitrocellulose/testing layer may be affixed to the membrane support 10.

As shown in FIG. 4, in one embodiment of the invention at one section ofthe test strip 7 is the sample site 16 to which the sample is to beapplied. This sample site 16 preferably has a sample pad 31 residing ontop of the testing layer 11, to which the sample is transferred. Thesample is preferably a bodily fluid. This fluid may be serum wholeblood, plasma, colostrum, milk, saliva, tears, or urine sample from ahuman or other animal species.

Downstream from the sample site 16 or sample pad 31 is the antigen, forwhich the serum is being tested. Upstream from the sample site 16 is thesite for the labeled analyte 12, which may reside on pad 13. The chaserelease buffer is added upstream from the labeled analyte after thesample has reached control line 22.

In a preferred embodiment of the invention (FIG. 5) for the testing ofallergens, the labeled analytes are anti-IgE antibodies 15 which havebeen attached to gold particles 14. The gold particles 14 attached tothe antibody 15 (or antigens in some instances, depending on the use ofthis lateral flow device) are preferably larger than 20 nm, morepreferably in the range of about 20 to 100 nm, and most preferably inthe range of 20 to 40 nm. The gold sol labeled antigens/antibodies 35are dried and deposited on the strip 7.

The metal sol particles to be used in accordance with the presentdisclosure may be prepared by coupling the analyte directly to the goldparticle. Additionally, the labeled component may be prepared bycoupling the analyte to the particle using a biotin/avidin linkage. Inthis latter regard, the substance may be biotinylated and the metalcontaining particle coated with an avidin compound. The biotin on theanalyte may then be reacted with the avidin compound on the particle tocouple the substance and the particle together. In another alternativeform of the disclosure, the labeled component may be prepared bycoupling the analyte to a carrier such as bovine serum albumin (BSA),keyhole lymphocyananin (KLH), or ovalbumin and using this to bind to themetal particles.

The metal sol particles to be used in accordance with the presentdisclosure may be prepared by methodologies which are well known. Forinstance, the preparation of gold sol particles is disclosed in anarticle by G. Frens, Nature, 241, 20-22 (1973). Additionally, the metalsol particles may be metal or metal compounds or polymer nuclei coatedwith metals or metal compounds, as described in U.S. Pat No. 4,313,734.Other methods well known in the art may be used to attach the analyte togold particles. The methods include but are not limited to covalentcoupling and hydrophobic bonding. The metal sol particles may be made ofplatinum, gold, silver, selenium, or copper or any number of metalcompounds which exhibit characteristic colors.

Similarly, the analyte does not necessarily have to be attached to ametal sol particle, but may instead be attached to dyed or fluorescentlabeled microparticles such as latex, polystyrene, dextran, silica,polycarbonate, methylmethacrylates and carbon. The metal sol particles,dyed or fluorescent labeled microparticles should be visible to thenaked eye or able to be read with an appropriate instrument(spectrophotometer, fluorescent reader, etc.).

There are a number of ways in which the gold labeled antigens 16 may bedeposited on the strip 7.

In yet another embodiment of the disclosure, the analytes may beattached to microspheres. This has the effect of increasing the numberof reactive sites (epitopes) in a given area. Analytes may be attachedto these alternate solid phases by various methodologies.

For instance, reactive microspheres (MX-Covaspheres.sup.R of diameter0.5 micrometers or 0.9 micrometers) purchased from Duke ScientificCorporation, Pal Alto, Calif. 94303, or other suppliers, may be used tocovalently attach analytes. The binding is at the amino groups of theprotein if covalent methodology is used. In addition, hydrophobic orelectrostatic domains in the protein may be used for passive coating. Asuspension of the spheres is mixed after sonication with theantigens/antibodies in water or in a phosphate buffer solution, afterwhich they are incubated at room temperature for 10-75 minutes. Themixture is then centrifuged and the pellets containing theantigen/antibody-linked microspheres are suspended in a buffercontaining 1-5% wt/volume bovine serum albumin (BSA) for 1 hour at roomtemperature. The BSA blocks any unreacted surfaces of the microspheres.After one more centrifugation, the spheres are resuspended in buffer(TBS with 5% BSA) and stored at 4 degrees C. before using.

The solid phase particles may comprise any one of known, waterdispersable particles, such as, the polystyrene latex particlesdisclosed in U.S. Pat. No. 3,088,875. Such solid phase materials simplyconsist of suspensions of small, water-insoluble particles to whichantigens/antibodies are able to bind. Suitable solid phase particles arealso disclosed, for example, in U.S. Pat. Nos. 4,184,849; 4,486,530; and4,636,479.

In another embodiment of the disclosure, the analytes may be attached tofluorescent microspheres or fluorescent microparticles. Said fluorescentmicroparticles may be purchased from Duke Scientific, Palo Alta, Calif.94303 and are listed as Green, Red, or Blue fluorescent 0.4 micronmicrospheres (Product Bulletin 93). They are also available fromMolecular Probes, Eugene, Oreg. 97402 and are listed as FluoroSpheres;Blue, Yellow-Green, Nile Red, Orange, Red, Crimson, Dark Red and Far Redin micron sizes from 0.03 to 5.0. Other manufactures also supplyfluorescent microspheres. Characteristically, fluorescent microspheresincorporate fluorescent dyes in the solid outer matrix or in theinternal volume of the microsphere. The fluorescent spheres aretypically detected by a fluorescent reader that excites molecules at onewavelength and detects the emission of fluorescent waves at anotherwavelength. For example, Molecular Probes Nile Red particles excite at526 nm at emit at 574 nm, the Far Red excites at 680 nm and emits at 720nm and the Blue excites at 365 nm and emits at 430 nm. In a lateral flowformat, detection of fluorescent microparticles requires the use of areflectance reader with an appropriate excitation source (HeNe, Argon,tungsten or diode laser) and an appropriate emission filter fordetection. Use of diode lasers allows for use of detection systems thatuse low cost lasers with detection above 600 nm. Most backgroundfluorescence is from molecules that emit fluorescence below 550 nm.

Fluorescent microspheres contain surface functional groups such ascarboxylate, sulfate and aldehyde groups, making them suitable forcovalent coupling of proteins and other amine containing biomolecules.In addition, sulfate, carboxyl and amidine microspheres are hydrophobicparticles that will passively absorb almost any protein or lectin.Coating is thus similar as for non fluorescent microspheres(MX-Covaspheres or other latex microparticles). A suspension of thefluorescent spheres is mixed after sonication with the antigens/antibodyin water or in a phosphate buffered solution, after which they areincubated at room temperature for 10-75 minutes. EDAC (solublecarbodiimide), succinimidyl esters and isothiocyanates as well as othercrosslinking agents may be used for covalent coupling of proteins andlectins to the microspheres. After the protein has attached to thesurface of the microparticles, the mixture is centrifuged and thepellets containing the antigen or antibody linked to the fluorescentmicroparticles are suspended in a buffer containing 1-5% bovine serumalbumin for one hour. After one more centrifugation, the spheres areresuspended in buffer (TBS with 5% BSA or other appropriate buffers) andstored at 4 degrees C. before use.

The solid phase particles useful in connection with the disclosure maycomprise, for example, particles of latex or of other support materialssuch as silica, agarose, glass, polyacrylamides, polymethylmethacrylates, carboxylate modified latex and Sepharose. Preferably, theparticles will vary in size from about 0.2 microns to about 10 microns.In particular, useful commercially available materials include 0.99micron carboxylate modified latex, cyanogen bromide activated Sepharosebeads (Sigma), fused silica particles (Ciba Coming, lot #6),isothiocyanate glass (Sigma), Reactogel 25DF (Pierce) andPolybead—carboxylate monodisperse microspheres. In accordance with thedisclosure, such particles may be coated with a layer of antigenscoupled thereto in a manner known per se in the art to present the solidphase component.

The Invention Thus Operates as Follows:

In one embodiment of the invention there are four binding sitespositioned at the site of the immobilized testing site. The firstbinding site 18 is preferably to bind IgM. The second binding site 19 ispreferably a site to bind IgA. The third binding site 20 is for thebinding of IgG and the fourth binding site 22 is for a control. Morespecifically, each binding site is in the form of a striped line alongthe width of the test strip opening 9. At the site of each binding site,there are anti-Ig immunoglobulins. For example, class specificantibodies are laid down on the test strip. For example, a goatanti-human IgM antibody is laid down at the first binding site 18, goatanti-human IgA antibody is laid down at the second site 19 and goatanti-human IgG antibody is laid down at the third binding site 20. Atthe control site there is immobilized a protein or substance containingsulfur residues that readily react with any colloidal gold compound. Itcan also be an antibody reactive with the proteins coated on the gold ormicroparticles surface. The antibodies reactive with IgM, IgA and IgGcan be from affinity purification of immune sera from goats, rabbits,donkeys, sheep, chickens or other animals. It may also be monoclonalantibodies directed against IgM, IgA and IgG. The antibodies used arespecific for the heavy chain portion of the IgM, IgA and IgG antibodies.Substances reactive with IgG (protein A, protein G, lentil lectin,jacalin, concanavilin A, mannan binding protein, wheat germ lectin,peanut lectin and avidchrom) may be substituted for the antibody to IgGor combined with said antibody.

In one embodiment, after the sample has been placed at the sampleopening 8, the sample migrates to the immobilized testing site, whereany appropriate reactions take place where the antibodies are reactivewith the site, thus forming a first complex. A chase release buffer isthen added upstream from, or at the site, of the labeled analytes. Thelabeled analytes are released and migrate to the site of the immobilizedfirst complex(es), where the labeled analytes bind to the firstcomplexes. When the labeled analytes (preferably gold labeled analytes)react with the first complex, a visible line emerges. The gold solcomplex. Migration continues and complexes are captured on the controlline of the test strip 7. Excess fluid is wicked into the absorbent pad17.

In another use and embodiment of the invention, it is disclosed here andshown in FIGS. 8 and 9 that immobilized antigens 201 of the binding site212 first be exposed to the antibodies 203 of the sample site 207. Theantibodies 203 (if present) of the sample react or bind to theimmobilized antigens 201 to form a first complex 204. Then, a Chaserelease buffer is added to or upstream from the labeled analytes 206,positioned upstream from the sample site 207 of the lateral flow assay208. The released labeled analytes 210 then migrate to the site 212 ofthe immobilized antigens 201, wherein the labeled analytes bind to thefirst complex 204, thereby forming a second complex 220 visible to thenaked eye. The labeled analyte 210 is preferably a gold labeled anti-IgEantibody.

In another embodiment of the invention as shown in FIGS. 7 and 8, thelabeled analytes 210 are incorporated, or, to describe the inventionmore succinctly, are absorbed into the actual test strip 100 at site106. Similarly, the binding site 104 can be incorporated into the teststrip 100, and the sample pad can similarly be eliminated, with directapplication of the sample on to the test strip downstream 105 from saidlabeled analytes.

There are a number of advantages to first exposing the sample (to betested) to the immobilized antigens to form a first complex beforeexposing the sample to the colorant. By allowing the “uninhibited”antibodies to bind to the antigens, there is less steric hindrance. Theunlabeled antibody molecules of the sample have a steriochemistry whichallows for easier binding between the samples antibodies and theappropriate antigens. Put another way, the unlabeled antibodies haveless physical or sterical interference and tertiary modifications,thereby allowing a more antibodies to bind to the antigens. The labeledanalyte does not mask any of the binding sites between the antigen andthe antigen specific antibodies, thus making it easier to read positivetest results.

As an alternative to a colorant, a fluorescent, radioactive, enzymatic,luminant or any other kind of marker can be used as a label that can beread/detected by the naked eye or by the appropriate equipment known inthe field.

As shown in FIG. 10, it should be noted that while antigens may beimmobilized on the lateral flow assay at the test site of the strip 305,antibodies or other specific bind agents may be laid down at the testsite 300 in those cases wherein the material to be tested is placeddownstream from the labeled analyte 301. In other words, the method (offorming a first complex between the sample and the test site “reagents”)may be used whether one is testing for different antibodies in the bloodto various antigens, or testing for any other biological or chemicalmaterial. Hence, depending on the nature of the test being performed, afirst complex may be formed between the sample and an antigen at thesite of testing zone, the sample and an antibody at the site of thetesting zone, or between the sample and any other material that willselectively bind to the sample being tested, with the sample placedbetween the labeling site and the immobilized testing site. As with thealternative embodiment of the present invention, the labeling site 301may be comprised of an absorbent pad 302. Similarly, the sample site 303may have a pad 304.

The outer plastic casing 400 (FIG. 11) has an opening 401 for theaddition of the chase release agent, and an opening 402 for the additionof the sample to be tested downstream from 401 (and downstream from theimmobilized analyte on the test strip) There is of course an opening 403to read the test results.

There are other embodiments for that may also be considered for theproposed lateral flow device.

In one instance, as shown in FIGS. 12 and 13, a sample is added in alateral flow device 500 through opening 501. The sample (assumingpositive results) will first bind to the immobilized antigens present onthe strip, thereby forming a first complex. After waiting a short time,the chase release buffer is added through the same opening 501 as thatof the sample. The volume of the sample added is preferably more thanthe amount of the chase release buffer; hence, in a preferable use ofthe invention, only the chase release buffer causes the gold reagent 603to migrate to the site of the testing zone 602 on the test strip 600,whereupon a second complex is formed, this time between the colorantreagent (gold reagent) and the first complex. For example, if an sampleis being tested for antibodies to specific allergen, a reaction willfirst form between the sample's IgE antibodies and the allergen on thetest strip, followed by a reaction between the gold labeled anti-IgEantibodies and the IgE-antigen complex(es). This method can be used totest for other antibodies, H. pylori, or any other appropriatesubstance.

In another embodiment of the device shown in FIG. 14, there is no needto have colorant or gold reagent residing on the test strip or in thetest device 700. The sample is added to the sample site 701 (which mayor may not have a sample pad) on test strip 700. After waiting until thesample has migrated to the control line 503 and has bound to theappropriate immobilized parts of the testing zone forming anyappropriate first complexes, a liquid gold reagent (or appropriatecolorant) is added through the same opening 500 as that of the sample.The liquid gold reagent will migrate to the site of the first complexesto form a second complex between the gold label reagent and the firstcomplex(es).

It should be noted that once the appropriate antibodies or analyte thatis being tested binds to the immobilized reagents, it will not bepossible for any stray gold labeled-reagent-analyte complexes to bind tothe testing site, because all or virtually all of the immobilizedbinding sites on the strip will have been occupied by the sample. Itshould further be note that virtually all of the sample migrates totesting site when first applied to the test strip in this alternativeembodiment.

In yet another embodiment of the invention shown in FIG. 15, chaserelease buffer can be added to the bottom section 801 of plastic casing800. Specifically, after sample has been added through sample openings804 and allowed to migrate to the control line 806 of the testing site805, chase release buffer is added to the openings 807 of the bottomsection thereby forming a reservoir, whereupon the top section 802 andthe bottom section 801 of the plastic casing 800 are pushed together sothat the reactions and flow process can be completed. Alternatively, anyreservoir containing chase release buffer can be used to dip the ends ofthe test strips to begin the flow of the colorant.

Alternatively, after the sample has been applied the bottom sections ofthe test strips can, be dipped in any container containing liquid goldreagent (or any other tagged reagent).

Once again, like the other processes, this device and process can beused to test for antibodies, antigens, allergens, H. pylori or anyanalyte or sample.

A kit for testing various biological substances may be formed for fielduse of the lateral flow device. The kit could comprise: a chase releasebuffer; at least one test strip (as described above), and a plasticcasing holding said at least one test strip in place, said plasticcasings having a sample opening in which to add the sample. Whereappropriate the kit can comprise the liquid gold reagents as describedabove in the various embodiments.

The problem of separating reactivities of antibody classes lies in the10 to 15 fold excess of IgG over IgA and IgM specific antibodyreactivity with analyte reaction sites. If the IgG is allowed to reactat the same time or rate as other classes of antibody, the IgG will maskmost if not all the analyte epitopes, thereby decreasing or eliminatingthe activity of the IgM and IgA class antibodies to the analyte.

To solve this problem, an IgG reacting substance (which can be, amongothers, protein A, protein G, an antibody to IgG, lentil lectin,jacalin, concanavilin A, mannan binding protein, wheat germ lectin,peanut lectin and avidchrom) is added to the sample pad in order tocomplex the IgG such that the molecular weight of the complex is greaterthan 1 million. This large complex travels sufficiently slower than IgA,IgM, and IgE, thereby allowing these antibodies to react prior to theIgG. After reacting with the colored solid phase, the various reactedcomplexes are captured specifically at three sites by the antibodies toIgM, IgA, and IgG, or a substance reactive with IgG (protein A, proteinG, lentil lectin, jacalin, concanavilin A, mannan binding protein, wheatgerm lectin, peanut lectin and avidchrom).

Similarly, when said sample site and/or sample pad are downstream fromsaid labeling site, said IgG reacting substance may be placed at thesample site and/or sample pad or downstream from said sample but beforethe test site.

For example, to determine whether a person has been exposed toHelicobacter pylori, or to determine if there has been successfultreatment of the disease, a serum sample is tested to determine whetherit contains antibodies to H. pylori. Assuming the use of gold labels, iflines appear at the binding sites for. IgG and weakly for IgM, thenthere is only a chronic condition present. If however, lines appear atthe binding sites for IgM and IgA, with or without IgG, then an activeor recent colonizing infection is occurring. The detection of IgA whencombined with a low serum pepsinogen level is associated with anincreased risk of gastric cancer.

This test can detect the presence of class specific antibodies reactivewith any bacteria, virus, fungus, irritant, or protein. Some, but notall of the analytes which can be detected using this method includeStreptococcus Group A, Streptococcus Group B, Mycobacterium,Mycobacterium tuberculosis, Mycoplasma, Chlamydiae, Rickettsiae, Herpesvirus, CMV, Hepatitis A, Hepatitis C, Hepatitis B, Influenza, HIV I, HIVII, HTLV I & II, Chagus, Toxoplasma, Helminh, Nematodes, autoimmuneantigens, antibodies to heat shock proteins, transplantation analytes,histocompatability analytes, and combinations thereof.

In another embodiment of the disclosure, this lateral flow assay can beused for the visual detection of allergen specific IgE antibodies inhuman or animal bodily fluids. In this assay the test serum reacts witha colorimetric (preferably gold) labeled anti-IgE antibody contained inthe colorimetric (preferably dried) gold pad 16. The resulting complextravels along the test strip to the line stripped allergen site 24. Atthe allergen site, there are a plurality of immobilized allergens 24.Indeed, the immunoassay can easily test for one or more differentallergens, preferably by one strip 25, two strips (25 and 26) ormultiple strips next to each other. Each strip can contain one or morespecific allergen lines. The common allergens which may be testedinclude but are not limited to pollens (Timothy, cultivated rye, birch,alder, hazelnut, mugwort, English plantain, ragweed, nettle, etc.), dustallergens (D. farinae, D. pteronyssinus, house dust), molds (Alternariatenuis, Aspergillus fum., Cladosporium, Penicillium not), animalepithelium (Cat epithelium, dog dander, horse dander, goose feathers)foods (dairy, cereals, nuts, seafoods, legumes and mixes thereof),inhalant mixes (pollen I (grasses), pollen II (weed/trees), animal mix,dust mix, mold mix) and combinations thereof The allergens areimmobilized on the test strip by the use of solubilizing agents such assugars and alcohols (sucrose, mannose, fructose, ethylene glycol,ethanol, methanol, glycerin, dextrans). The use of sugars and alcoholsunfolds the allergen protein tertiary structure such that morehydrophobic domains are exposed allowing greater binding to themembrane. In addition, protein to protein aggregation is reduced throughsolubilization allowing individual protein molecules to bind to thenitrocellulose or nylon membrane.

It should be noted that, in addition to the use of sugars and alcohols,the protein molecules or reagents may be immobilized on the test stripby the use of other materials, including but not limited to fats, gels,simple buffers, specially designed molecules.

Assuming there is a reaction between the complexes of gold labeled antiIgE antibody and the sample containing IgE antibody and the allergens, ared line will appear at the site of the allergen when there is apositive response. The assay validity is demonstrated by the appearanceof a red colored line in the positive control region of the membrane.The positive control is a protein or substance containing sulfurresidues that readily react with any colloidal gold compound. It canalso be an antibody reactive with the proteins coated on the gold or inthe sample. Since the gold or microparticles conjugate is in excess,sufficient conjugate is available to react with the control line.

Many modifications and variations of the present disclosure are possiblein light of the above teachings. It is, therefore, to be understoodwithin the scope of the appended claims the disclosure may be protectedotherwise than as specifically described.

Downstream from the sample site 16 or sample pad 31 is the antigen, forwhich the serum is being tested. Upstream from the sample site 16 is thesite for the labeled analyte 12, which may reside on pad 13. The chaserelease buffer is added upstream from the labeled analyte after thesample has [been at 16] reached control line 22.

1. A lateral flow immunoassay device for detecting immune reactants,said device comprising: at least one test strip, said at least one teststrip comprising: a) a sample site for applying a sample comprisingantibodies; b) a plurality of reaction sites downstream from said samplesite, each said reaction site containing a different allergen such thatwhen IgE antibodies come in contact with an allergen to which the IgEantibodies react, a first complex is formed; c) a colorimetric labeledanalyte site comprising a colorimetric labeled anti-IgE antibody, saidcolorimetric labeling site positioned upstream from said sample site;and d) a location on said test strip to add a chase release buffer forthe release of said labeled analyte; wherein said colorimetric labeledanti-IgE antibody, released by addition of said chase release buffer,migrates to the site of the at least one first complex, forming acolorimetric labeled Anti-IgE antibody—IgE antibody-allergen complex. 2.The lateral flow immunoassay device of claim 1, wherein said immunoassaycomprises at least two test strips.
 3. The lateral flow immunoassaydevice of claim 1, wherein said allergens are selected from the groupsconsisting of pollens, dust mite allergens, molds, animal epithelium,foods, allergen mixes, and combinations thereof.
 4. The lateral flowimmunoassay of claim 1, wherein said at least one solubilizing agent toimmobilize any said allergen, said at least one solubilizing agent beingpresent in an amount such that said allergen protein tertiary structureunfolds to allow for greater binding of said antigen to said test strip,wherein, said solubilizing agent is selected from the group consistingof sugars and alcohol.
 5. The lateral flow immunoassay of claim 1,wherein said at least one solubilizing agent is selected from the groupconsisting of sucrose, mannose, fructose, ethylene glycol, ethanol,methanol, glycerin and dextrans.
 6. The lateral flow immunoassay ofclaim 1, further comprising a solubilizing agent wherein said at leastone solubilizing agent is a sugar.
 7. The lateral flow immunoassay ofclaim 1, wherein said at least one solubilizing agent is an alcohol. 8.The lateral flow immunoassay device of claim 6, wherein said sugar isselected from the group consisting of sucrose, mannose, fructose,dextran, and combinations thereof.
 9. The lateral flow immunoassaydevice of claim 7, wherein said wherein said alcohol is selected fromthe group consisting of ethylene glycol, glycerol, ethanol, methanol,and combinations thereof.
 10. A method of detecting immune reactants,comprising: a) placing a sample containing antibodies on a lateral flowimmunoassay device, said device comprising: at lease one test strip,said at least one test strip comprising: i) a sample site for applying asample comprising antibodies; ii) a plurality of reaction sitesdownstream from said sample site, each said reaction site containing adifferent allergen such that when IgE antibodies come in contact with anantigen to which the IgE antibodies react, a first complex is formed;iii) a colorimetric labeled analyte site comprising a colorimetriclabeled anti-IgE antibody, said colorimetric labeling site positionedupstream from said sample site; and iv) a location on said test strip toadd a chase release buffer for the release of said labeled analyte; b)allowing said test sample to migrate from the sample site to thereaction sites; c) adding said chase release buffer; and d) reading saidlateral flow immunoassay device, wherein a colored line, indicating apositive response will develop when said colorimetric labeled anti-IgEantibodies come in contact with an IgE antibody which has reacted withan antigen.
 11. The method of claim 10, wherein said allergens areselected from the group consisting of pollens, dust mite allergens,molds, animal epithelium, foods, allergen mixes, and combinationsthereof.
 12. A lateral flow assay device for detecting immune reactants,said device comprising: at least one test strip, said at least one teststrip comprising: a) a sample site for applying a sample comprising atleast one analyte for which the device is designed to detect; b) atleast one reactant downstream from said sample site, such that when saidat least one analyte comes in contact with said reactant, a firstcomplex is formed; c) a colorimetric label site comprising acolorimetric label, said colorimetric labeling site positioned upstreamfrom said sample site; said colorimetric label capable of reacting orattaching to the analyte, said colorimetric labeled having the abilityto be released from said colorimetric label site by a chase releasebuffer; and d) a location on said test strip to add said chase releasebuffer, wherein said colorimetric label, released by addition of saidchase release buffer, migrates to the site of the at least one firstcomplex, forming a colorimetric labeled analyte reactant.
 13. A lateralflow immunoassay device kit for detecting immune reactants, said kitcomprising: I) a chase release buffer; II) at least one test strip, saidat least one test strip comprising: a) a sample site for applying asample comprising antibodies; b) a plurality of reaction sitesdownstream from said sample site, each said reaction site containing adifferent allergen such that when IgE antibodies come in contact with anallergen to which the IgE antibodies react, a first complex is formed; acolorimetric site for labeling the first complex, forming a colorimetriclabeled Anti-IgE antibody-IgE antibody-allergen-complex, saidcolorimetric labeling site comprising a colorimetric labeled anti-IgEantibody, said colorimetric labeling site positioned upstream from saidsample site, said colorimetric labeled anti-IgE antibody being releasedby said chase release buffer; and d) a location on said test strip toadd said chase release buffer; wherein said allergens are immobilized tosaid test strip using at least one solubilizing agent, said at least onesolubilizing agent being present in an amount such that said allergenprotein tertiary structure unfolds to allow for greater binding of saidallergen to said test strip, wherein said at least one solubilizingagent is selected from the group consisting of sugars and alcohols; andIII) a plastic casing holding said at least one test strip in place,said plastic casings having a) a sample opening in which to add thesample: b) a second opening in which to add the chase release buffer,said second opening positioned upstream from said sample opening andupstream from said colorimetric site; and c) a third opening in which toview the labeling sites after the sample and the chase release bufferhave been added.
 14. The lateral flow immunoassay device kit of claim13, wherein said immunoassay comprises at least two test strips.
 15. Thelateral flow immunoassay device kit of claim 13, wherein said allergensare selected from the groups consisting of pollens, dust mite allergens,molds, animal epithelium, foods, allergen mixes, and combinationsthereof.
 16. The lateral flow immunoassay device kit of claim 13,wherein said at least one solubilizing agent to immobilize any saidallergen is present in an amount such that said allergen proteintertiary structure unfolds to allow for greater binding of said antigento said test strip, wherein, said solubilizing agent is selected fromthe group consisting of sugars and alcohol.
 17. The lateral flowimmunoassay device kit of claim 13, wherein said at least onesolubilizing agent is selected from the group consisting of sucrose,mannose, fructose, ethylene glycol, ethanol, methanol, glycerin anddextrans.
 18. The lateral flow immunoassay device kit of claim 13,wherein said alcohol of said solubilizing agent is selected from thegroup consisting of ethylene glycol, glycerol, ethanol, methanol, andcombinations thereof.